All-trans-retinoic acid and 11-cis-retinal play crucial roles in such processes as development, immunity and vision. They are biosynthetically derived from all-trans-retinol or vitamin A. The enzyme lecithin:retinol acyltransferase (LRAT) plays a pivotal role in the absorption of retinol from the diet and storage of retinol in specialized cells in the liver and eye. In humans, natural mutations in the LRAT gene lead to severe retinal dystrophy known as Leber Congenital Amaurosis, and disruption of the Lrat gene in Lrat-/- mice leads to severely impaired retinol storage and blindness.

Delivery of retinol to specific target tissues is important in several therapeutic settings including vitamin A deficiency, certain eye diseases and cancer. LRAT may offer promise as a target for therapies aimed at affecting retinoid levels and/or metabolism in target tissues, but achieving this goal will require knowledge of its structure and mechanism. LRAT is a membrane protein that processes very lipophilic substrates. Its mechanism is poorly understood and it lacks sequence similarity to other known enzymes. Using N-linked glycosylation scanning and protease protection assays we showed that LRAT is localized to the membrane of the endoplasmic reticulum (ER) where it assumes a single-spanning membrane topology with an N-cytoplasmic/C-lumenal orientation. The C-terminal domain of LRAT is essential for both catalytic activity and ER membrane targeting, whereas the N-terminal hydrophobic region is not.

This COBRE Pilot Project has two specific aims.

Aim 1. Eludicate the mechanistic details of LRAT and its interactions with its accessory protein CRBP (cellular retinol binding protein). These studies will be conducted in conjunction with the Protein Production Group of the COBRE Center.

Aim 2. We will investigate the structure of LRAT by X-ray crystallography. High-resolution data collection and model building will be conducted in collaboration with the COBRE Protein Structure Core Laboratory.